The long-term objective of this proposal is to develop tumor-targeted TCR-IL15 fusion proteins as immunotherapeutics for treating cancers. Previous proof-of-concept studies with TCR-IL2 and TCR-IgG1 fusion proteins in mouse tumor efficacy models demonstrated the feasibility of this approach and have led to the clinical evaluation of one of these fusion proteins in patients with metastatic cancer. However, recent research suggests that IL-15 is capable of enhancing tumor immunosurveillance without the immunosuppressive activity associated with IL-2-mediated responses. To evaluate whether tumor-targeted IL15 protein exhibits superior antitumor activity, we first created two soluble single-chain (sc) TCR-IL15 constructs: one (referred to as c264scTCR) specific for a peptide derived from the tumor associated protein p53 presented in the context of HLA-A2.1 and a second (OT1scTCR) specific to the well-characterized ovalbumin peptide/Kb antigen. We then constructed scTCR fusions with the soluble domain of the IL-15 receptor a subunit which has the capability of stably binding to IL-15, allowing generation of heterodimeric scTCR-IL15/scTCR-IL15Ra complexes with increased antigen binding activity. We then further introduced novel IL-15 mutations with super-agonist and antagonist activity into these fusion proteins. We hypothesize that these fusion proteins and protein complexes may have several advantages as immunotherapeutics by providing improved bioactivity and an increased half-life compared to IL-15 alone as well as targeting IL-15-dependent cytotoxic immune responses to the tumor site through multivalent binding with the TCR-specific antigen. With the availability of these reagents, we are in the position to evaluate their antitumor activities and safety profiles in relevant animal models. Thus, the overall objectives of Phase I and II of this proposal are to evaluate the antitumor efficacy, and to determine the mechanism-of-action of these scTCR-IL15 fusions in murine and human xenograft tumor models, complete preclinical pharmacokinetic, pharmacodynamic and toxicology studies and optimize production and purification methods necessary to advance the c264scTCR-IL15 fusions into clinical trials. Since p53 is overexpressed on around 50% of all tumors, demonstration of the safety and antitumor efficacy of c264scTCR-IL15 fusions in the clinic could be of great benefit to a broad population of cancer patients. The following Phase I specific aims will be pursued to evaluate of the scTCR-IL15 fusion proteins as the initial efforts toward this project's goals: 1) complete the in vitro characterization of scTCR-IL15 fusions and scTCR-IL15/scTCR-IL15Ra complexes, 2) evaluate the antitumor efficacy of tumor- specific (and control) scTCR-IL15 fusions in mouse EG7 tumor and xenograft p53-positive human tumor models, and 3) determine pharmacokinetics and safety profile of scTCR-IL15 fusions and scTCR-IL15/scTCR-IL15Ra complexes in HLA-A2-transgenic mice. PUBLIC HEALTH RELEVANCE: The goal of the proposed research is to examine the safety and efficacy of a novel tumor-targeted immunotherapeutic in animal models. If successful, this compound could potentially have antitumor activity against a broad number of cancers that expresses a common tumor-associated biomarker. Ultimately, this approach may provide a safer and more effective treatment for patients with cancer than the currently approved immunotherapies.